作者要感谢伊丽莎白·布兰查德对她的技术援助。本报告中的调查结果和结论是这些作者的观点，并不一定代表美国疾病控制和预防中心的意见。

1。培养的CALU-3细胞用于在Transwell文化 安全对策：执行使用无菌培养技术，生物安全柜中的所有程序。 <ol><li>解冻细胞冷冻储藏： <ol><li>准备的Eagle氏最低必需培养基，含有20％热灭活的牛胎儿血清（FBS），0.1mM非必需氨基酸，2mM的L-谷氨酰胺，和10毫摩尔HEPES PH值7.4（EMEM-20％+ S）。无菌过滤器制备的介质，使用0.2微米的孔隙大小的过滤器，并在水浴中温至37℃。为了更反映了地气道上皮细胞环境，中不中添加抗生素或抗真菌药。 </li><li> CALU-3细胞迅速（&lt;1分钟），在37℃水浴解冻冷冻离心管。 </li><li>将解冻的细胞转移到50ml无菌锥形管中，并添加30毫升温热的EMEM-20％+ S中</li><li>通过离心沉淀细胞5分钟在1,000-1,200×克，无需冷藏，低速制动器速度。 </li><li>轻轻地倒出上清液，并重新悬浮的细胞在5毫升温热的EMEM-20％+ S中，轻轻移液管向上和向下。种子2〜5×10 6个细胞每孔到6孔组织培养板中，孵育在37℃，7％CO 2中，在空气氛围中，直到细胞是75％ - 80％汇合。每天检查，可能需要长达7天。每3-4天吸介质的细胞，取而代之的是新鲜的EMEM - 20％+ S. </li></ol></li><li>继代培养细胞： <ol><li>温暖的0.05％胰蛋白酶 - 0.02％EDTA在37°C水浴。从细胞中吸液的介质，和洗涤细胞一次，温热的0.05％胰蛋白酶 - 0.02％的EDTA，以除去过量的介质和血清。取出洗净，加热胰蛋白酶，每孔加入1毫升的细胞。 </li><li>孵育在37℃，7％CO 2和显示器在显微镜下，直到至少75％的细胞脱离以及（s）的组织培养板的每5分钟。这可能需要5到30分钟。 </li><li>在EMEM-20％+ S洗涤细胞以除去过量的胰蛋白酶。胰蛋白酶处理细胞转移到50ml无菌锥形管。从一个6孔培养板中的细胞的多口井可以汇集到一个50毫升无菌锥形管。加入30毫升的EMEM-20％+ S和颗粒的细胞通过离心分离在1,000-1,200×克，无需冷藏，与制动速度低。 </li><li>轻轻地倒出上清液，在新鲜EMEM-20％+ S和重悬细胞，轻轻移液管向上和向下。使用步骤1.2.1到1.2.3以上trypsinize细胞的继续传代培养细胞培养皿中描述的步骤1.2.5至1.2.7时，他们是在75 - 80％汇合。 </li><li>继代培养从一个以及一个6孔板一个T-25厘米2烧瓶中，终体积为5毫升的EMEM + S-20％，播种在0.5〜1×10 6个细胞/瓶之间。 </li><li>每使用5毫升暖胰蛋白酶T-25厘米2烧瓶中分离细胞，传代培养从一个T-25cm 2的烧瓶中的一个之间的T-75 cm 2的烧瓶中，终体积为10毫升的EMEM-20％+ S，播种1〜5×10 6个细胞/瓶。 </li><li>每使用8毫升暖胰蛋白酶T-75厘米2烧瓶从T-75厘米2烧瓶2 T-75厘米2瓶中分离细胞，传代培养。为了获得最佳的细胞生长，不大于T-75 cm 2或传代培养的比率大于1父烧瓶中：3个新的培养瓶中到烧瓶中的传代培养细胞。 </li></ol></li><li>一旦传代培养细胞已被完全删除和替换介质每2到3天，直到它们达到75-90％汇合。细胞可能需要长达3周达到75-90％汇合。对于产生的极化文化，亚文化群增长超过90％融合成单层细胞，才最佳。 </li><li>继续，直到足够的细胞的传代培养细胞生长到Transwell小室的所需数量的种子。每个Transwell小插入需要2×10 5细胞。对于最佳性能产生极化文化，使用的细胞都发生了不超过10个亚文化。 </li></ol> 2。成长偏光CALU-3液体覆盖的文化（LCC） 安全对策：执行使用无菌培养技术，生物安全柜中的所有程序。 <ol><li>准备EMEM中含有10％FBS的EMEM-10（％），在水浴中温到37℃。 </li><li>准备一个24孔Transwell小板的播种。用无菌镊子，将Transwell小室从内部到外部的板行插入膜不接触。细胞应该只传代培养成井外板的行。为了防止气泡引进到基底室，加600μl的EMEM 10％，每一个对每个车厢壁和倾斜吸管，慢慢地释放介质入井。 </li><li>从T-75厘米2组织中分离细胞。 Subculture烧瓶使用温热的胰蛋白酶，并在30毫升的EMEM-20％+ S每每2烧瓶中的胰蛋白酶消化细胞，如步骤1.2中描述的清洗。 </li><li>彻底洗过的细胞悬浮在5毫升的EMEM-10％，轻轻移液管向上和向下。 </li><li>确定的可行和总细胞数台盼蓝排除法。继续执行仅在80％ - 90％是可行的。 </li><li>在50ml的无菌锥形管中，稀释细胞浓度为2×10 6个活细胞/ ml的EMEM-10％。 </li><li>添加单元格的顶舱的每个Transwell小。至水井A1和D1，这将是无细胞的控制井，即空白，加入100μl的EMEM-10％无细胞。对于每个剩余的井，再悬浮CALU-3细胞中加入100μl，轻轻垂钓吸移管对内部插入壁的引导通道，并慢慢地释放细胞。请勿触摸移液管插入膜。 CALU-3细胞，以维持均匀的悬浮液，轻轻搅拌细胞悬浮液在此播种一步。 </li><li>孵育Transwell小板在37℃，7％CO 2中 ，在空气氛围。现浇板的孵化器，物理干扰将是最小的。为了提高效率的偏振，不要堆放板彼此的顶部上。 </li><li>为了保持文化，直到两极化，在实验中，使用完全更换介质中的Transwell小室内，如以下步骤2.10至2.13。中应完全取代传代培养后三日内到Transwell小室内，然后在周期交替之间的第四和第三的一天后，以前的喂养，直至细胞完全极化。 </li><li>暖的EMEM在水浴中的10％〜37℃。 </li><li>轻轻吸出培养基，Transwell小室。用毛细管的吸液管连接到用温和的真空的真空陷阱，或与标准1毫升移液管，从无细胞井A1和D1的吸出培养基，然后从种子井，先除去心尖介质从所有孔中，和t母鸡删除所有井的基底介质。吸介质时，请勿触摸插入膜。 </li><li>加入200μl的EMEM-10％的无细胞井A1和D1的根尖室中，然后到种子井，指挥到根尖使用插入侧引导移液管尖的舱室中的介质。不要添加介质直接到细胞，而不要触摸插入膜。 </li><li>加入600微升EMEM-10％的基底舱室。为了防止引进气泡到基底车厢，加中每一个对每个车厢壁和倾斜吸管，慢慢地释放介质入井。 </li></ol> 3。的CALU-3 LCC抗性发展评价 安全对策：执行使用无菌培养技术，生物安全柜中的所有程序。 <ol><li>评估跨皮电阻（TEER）的CALU-3 LCC 30分钟后，介质变化;每个媒体的变化后，如果需要的话，可以进行评价。在无菌条件下进行测量。开始与无细胞控制孔A1和D1（空白）的测量，以获得基线测量，并继续测量各孔。 <ol><li>在无菌条件下，转移到50毫升离心管中，含有70％的乙醇在无菌水中的STX2电极，并灭菌15分钟。 </li><li>校准和测试用于voltohmmeter根据制造商的指示。 </li><li>从乙醇，空气干燥5-10秒，取出电极并冲洗电极用无菌的EMEM-10％。 </li><li>设置的voltohmmeter的模式开关的电阻设置，并关闭电源。 </li><li>轻轻将电极允许进入一个Transwell培养的基底室的3个港口之一。将电极较长的前置只需轻轻接触外口井的底部，并保持垂直，和较短的铅是根尖室在组织培养基中，不接触插入膜。 </li><li>按下“测量R”按钮，然后等待读数稳定。重复为每个孔的其他2个端口，并从所有三个端口，总共三个测量每孔中的测量记录。继续来测量电阻的单元格的所有油井。 </li><li>通过在70％乙醇中浸泡5-10分钟，在无菌蒸馏水2 O漂洗，干燥彻底清洁电极。存放在原来的容器中。 </li><li>计算各孔的电阻，利用公式1。 Ω 实际值 =Ω 样品 - Ω 空白 ， 式（1） Ω 样品是从种子好，Ω 空白的平均测量值是从2井，A1和D1，平均测量包含插入和中等的，但没有细胞。 </li><li>计算单位面积的电阻，使用方程TION 2。 Ω 实际 ×有效膜面积=Ω×cm 2时 ， 方程2 其中的有效膜面积为0.33厘米2的24孔Transwell小室。 </li></ol></li><li>验证的两极分化进行二次检测，测量被动荧光素钠扩散心尖和基底车厢之间，一至三Transwell小文化。用于荧光素钠测定的井应该被丢弃后立即测定完成。 <ol><li>准备非荧光的缓冲液（118 mM氯化钠，4.75 mM KCl中，2.53毫摩尔的CaCl 2 .2 H 2 O，2.44 mM的用MgSO 4，1.19毫摩尔KH 2 PO 4，在无菌水中的25mM的NaHCO 3，用0.45μm的过滤装置的无菌过滤器）。准备荧光素钠在1 mg / ml的非荧光的缓冲液，无菌过滤器，包裹在铝箔中，并储存在4℃，避光。温至室温temperatu的重新使用前。 </li><li>电阻测量完成后，小心地取出介质Transwell小文化的一至三个独立的基底和根尖车厢。 </li><li>冲洗Transwell小文化。轻轻地添加600微升的室温无菌的Dulbecco PBS（D-PBS），基底舱室和100μl无菌D-PBS室温到根尖舱室。 </li><li>轻轻地取出D-PBS来自水井。加入600μl无菌无荧光缓冲的基底车厢。加入100微升无菌1 mg / ml时，荧光素钠的根尖的舱室。 </li><li>板在37℃下孵育1小时。 CO 2的温育过程中不需要。 </li><li>在孵化过程中，准备荧光素钠的标准曲线范围为20μg/ ml和0微克/毫升，使用无荧光缓冲液稀释荧光素钠。准备至少为8个不同浓度的荧光素钠，每个至少为600微升，最终体积。保持避光，直到准备测定吸光度标准曲线稀释。 </li><li>转移非荧光的缓冲区的样本从基底每个测试Transwell小室中，以用于分析一个单独的干净的试管。删除的的测试Transwells用于检查被动荧光素钠的扩散板和丢弃。返回板与其余的Transwell小室内的孵化器。 </li><li> 100μl的一式三份的每个标准溶液放置到一个96孔的平底板。 </li><li>准备三个每个基底样品稀释液（1:2，1:20和1:50），在非荧光缓冲液中，并添加100μl的每个未稀释样品的各稀释，一式两份，将在96孔平底板。 </li><li>测量样品在ELISA板读数器在486纳米或490 nm处的吸光度。 </li><li>通过比较样品的吸光度的吸光度值对钠荧光日确定浓度的荧光素钠在基底室andard曲线。 </li></ol></li></ol> 4。呼吸道病毒感染极化的CALU 3 LCC 安全措施：执行生物安全柜中的所有程序用无菌培养技术，病毒在生物安全水平适当。 <ol><li>稀释，使所需的接种物在无血清的EMEM病毒将在100μl。 </li><li>洗涤细胞，在无血清的EMEM。轻轻吸出培养基，所有的孔，消除根尖介质，然后基底介质。用毛细管的吸液管连接到用温和的真空的真空陷阱，或与一个标准1毫升的移液管，从无细胞井A1和D1的吸出培养基，然后从井种子，第一除去根尖介质从所有孔中，然后除去所有井的基底介质。吸介质时，请勿触摸插入膜。 <ol><li>添加100μl的无血清的EMEM，无细胞的孔A1和D1心尖室中，然后以吨他种子井，指挥介质到根尖使用插入侧引导移液管尖的舱室。不要添加介质直接对细胞，而不要触摸插入膜。 </li><li>加入600μl的无血清的EMEM基底舱室。垂钓吸移管对的隔室的壁和缓慢释放入井介质，将介质每个Transwell小。 </li><li>轻轻地从井中吸取的无血清培养基。 </li></ol></li><li>与未感染或感染的模拟井开始，添加适当的的病毒稀释，根尖车厢内的Transwell小室内。对于未感染的井，添加100μl的无血清的EMEM根尖室中适当的Transwell小室内，用于模拟感染的井，加入100μl的无病毒的制剂稀释于无血清的EMEM心尖室适当的Transwell小室内，并病毒感染，稀释在无血清的EMEM病毒和根尖的室中适当的Transwell小室内加入100μl。 </li><li>加入600μl的无血清的EMEM的所有基底舱室。 </li><li>孵育在37℃，7％CO 2的 2小时。 </li><li>吸出培养基，首先从感染和模拟感染井，井，然后从被感染的井。取出根尖的上清液第一，其次是基底上清。 </li><li>替换介质与200μl的EMEM-10％在心尖车厢和600微升的EMEM-10％在基底舱室。 </li></ol>

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T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Severe+acute+respiratory+syndrome+(SARS)+coronavirus-induced+lung+epithelial+cytokines+exacerbate+SARS+pathogenesis+by+modulating+intrinsic+functions+of+monocyte-derived+macrophages+and+dendritic+cells.">Severe acute respiratory syndrome (SARS) coronavirus-induced lung epithelial cytokines exacerbate SARS pathogenesis by modulating intrinsic functions of monocyte-derived macrophages and dendritic cells.</a> Journal of virology. 83, 3039-3048 (2009).</li><li>Yoshikawa, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+innate+immune+responses+of+human+bronchial+epithelial+cells+to+severe+acute+respiratory+syndrome-associated+coronavirus+infection.">Dynamic innate immune responses of human bronchial epithelial cells to severe acute respiratory syndrome-associated coronavirus infection.</a> PloS one. 5, e8729 (2010).</li><li>Hsu, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Critical+role+of+constitutive+type+I+interferon+response+in+bronchial+epithelial+cell+to+influenza+infection.">Critical role of constitutive type I interferon response in bronchial epithelial cell to influenza infection.</a> PloS one. 7, e32947 (2012).</li><li>Harcourt, J. L., Caidi, H., Anderson, L. J., Haynes, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+the+Calu-3+cell+line+as+a+model+of+in+vitro+respiratory+syncytial+virus+infection.">Evaluation of the Calu-3 cell line as a model of in vitro respiratory syncytial virus infection.</a> Journal of virological methods. 174, 144-149 (2011).</li><li>Zhang, L., Peeples, M. E., Boucher, R. C., Collins, P. L., Pickles, R. 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没有利益冲突的声明。

当建立CALU-3中的LCC的Transwell小室的细胞可能不极化在所有，或可能不完全分化，定义由TEER≥1,000Ω×cm 2和≤1％荧光素钠染料心尖和基底舱室之间达到平衡。此外，在LCC的CALU-3细胞可以充分分化，但，TEER可能是不一致的测量之间的。虽然从日常的TEER的测量的CALU-3 LCC的波动是正常的，一旦完全偏振光，TEER的剧烈波动没有预计到的文化自然随着年龄的下降，这可能是短短的5个星期或长达12周后播种。 CALU-3 LCC极化的能力部分地取决于细胞如何维持并传代培养在Transwell小室系统使用前。超出90％汇合的单层生长的细胞，具有在传代培养过程中，已传代培养从冷冻库存中的10倍以上，或没有连接提供一个定期的新鲜培养基上是不太可能完全分化，并，任何两极分化很可能迅速下降。也可能是由于不完整或完全没有极化使用的CALU-3 LCC的Transwell小室内的材料和孔径的变化，和很多很多类似的组成和孔径的Transwell小室内的变化也可能会影响极化。较大的孔径大小可以让CALU增长通过Transwell小室膜到基底外侧室，防止文化偏光。偏振情况下也可能是由于细菌的生长，表明云培养基，从而导致随后的崩溃CALU-3细胞之间的紧密连接。 可变TEER测量CALU-3 LCC的原因可能是由机械CALU-3 LCC细胞单层，插入，或在板本身的中断。中的变化和TEER测量应不吸头或电DE导致接触的细胞。在执行这些操作时，应注意避免气泡进入的顶端和基底室，这将破坏的能力，以检测电阻的voltohmmeter。可能也限制了蛋白质积聚在电极引线的能力voltohmmeter检测电阻的文化，实际上是极化。此积聚可轻轻打磨除去，或者可以通过更换电极校正。 一旦CALU-3 LCC完全分化和TEER不再增加，CALU-3 LCC是准备用于在主机肺上皮细胞的反应，呼吸道感染为特征的体外模型。该系统能够更好地表征方向的反应相比，传统上用来研究呼吸道病原体，如A549和HEp-2细胞的单层培养的肺癌细胞株的病原体，CALU-3 LCC北额外的优点纳克更快速的发展，越来越容易获得，并且不太昂贵的产生不是主要的，两极化，差异化的NHBE。 NHBE，极化CALU-3表现出紧密连接形成，并产生粘蛋白。然而，不像NHBE，偏振光CALU-3细胞不分化成层的基底细胞和纤毛柱状上皮细胞，以及几偏振光CALU-3细胞培养纤毛状突起19。因此，尽管用于研究极化反应的呼吸道上皮细胞呼吸侮辱，偏光CALU-3 LCC是不是一个理想的模型来研究气道呼吸的侮辱和伤害的开发或改造。 体外培养细胞的粘液生产可能会影响细胞的感染性，感染病毒和病毒传播中的极化模型以及释放，极化CALU LCC和两极化，差异化的NHBE尚未见报道的粘液生产之间的直接比较。 A549和HEp-2细胞重新较易培养比CALU-3细胞，然而，不像CALU-3 LCC，它们不形成偏振光文化Transwell小室上生长时， 在体外研究极化上皮细胞呼吸道病毒感染的响应，并因此是不理想的模型。

<table border="1"><tbody><tr><td></td><td></td><td></td><td> 试剂 </td></tr><tr><td> CALU-3 </td><td> ATCC </td><td> HTB-55 </td><td></td></tr><tr><td> 0.05％胰蛋白酶 - 0.02％EDTA的</td><td> Gibco / Invitrogen公司</td><td> 25300 </td><td></td></tr><tr><td>鹰的基本培养基（EMEM） </td><td> Gibco / Invitrogen公司</td><td> 07-00100DK </td><td></td></tr><tr><td>牛胎儿血清（FBS）的</td><td>胎牛血清</td><td> SH30070.03 </td><td>热灭活，56°C，30分钟</td></tr><tr><td>非必需氨基酸100X中（10mM） </td><td> Gibco / Invitrogen公司</td><td> 11140 </td><td>在黑暗中在4℃下储存</td></tr><tr><td> L-谷氨酰胺</td><td> Gibco / Invitrogen公司</td><td> 25030 </td><td></td></tr><tr><td> HEPES </td><td> Gibco / Invitrogen公司</td><td> 15630 </td><td></td></tr><tr><td> EMEM-10％FBS（EMEM-10％） </td><td></td><td></td><td>补编10％血清，无菌过滤器与热灭活FBS的EMEM </td></tr><tr><td> EMEM-20％FBS +补充剂（EMEM-20％+ S）的</td><td></td><td></td><td>最终浓度补编EMEM：热灭活FBS，20％; 1X氨基酸，2mM的L-谷氨酰胺，10mM的HEPES，无菌过滤</td></tr><tr><td> 24孔Transwell小板</td><td>康宁深圳科诗特</td><td> 3472 </td><td>孔径为3μm，聚酯</td></tr><tr><td>台盼蓝</td><td> Gibco / Invitrogen公司</td><td> 15250 </td><td></td></tr><tr><td>乙醇</td><td>西格玛</td><td> E7023 </td><td>准备70％使用无菌蒸馏水2 O </td></tr><tr><td>的Dulbecco PBS（D-PBS） </td><td> Invitrogen公司</td><td> 14040 </td><td></td></tr><tr><td>非荧光的缓冲液</td><td></td><td></td><td> 118毫米的NaCl;4.75 mM KCl中，2.53 mM的的CaCl 2×H 2 O; 2.44 mM的用MgSO 4; 1.19 mM的KH 2 PO 4，在无菌水中的25mM的NaHCO 3;无菌过滤器</td></tr><tr><td>荧光素钠</td><td>西格玛</td><td> 6377 </td><td> 1毫克/毫升在无菌非荧光的缓冲区;无菌过滤器，保护从光;储存于4℃下高达6个月</td></tr><tr><td></td><td></td><td></td><td> 设备 </td></tr><tr><td> Voltohmmeter </td><td>世界精密仪器</td><td></td><td></td></tr><tr><td> STX2电极</td><td>世界精密仪器</td><td></td><td></td></tr><tr><td> ELISA板读数器</td><td></td><td></td><td>能够测量的486或490 </td></tr></tbody></table>

establishing a liquid-covered culture of polarized human airway epithelial calu-3 cells to study host cell response to respiratory pathogens in vitro

气道上皮细胞的顶端和基底表面展示了接触病原体在体内的定向反应。因此，理想的体外模型研究细胞对呼吸道病原体的分化，形成心尖和基底表面。这样的模式是有区别的正常人类支气管上皮细胞（NHBE）。然而，这种系统需要肺组织样本，专业知识分离和培养从组织中的上皮细胞，和时间，以产生一个空气 - 液体界面的文化。 CALU-3细胞，来自一个人支气管腺癌，是研究近端呼吸道上皮细胞的响应呼吸侮辱1，药理化合物2-6，和7-9的细菌和病毒病原体的，包括流感病毒，鼻病毒的替代模式与严重急性呼吸系统综合症-相关的冠状10-14。最近，WE展现CALU-3细胞很容易受到呼吸道合胞病毒（RSV）感染在与NHBE 15,16一致的方式。在这里，我们建立了详细的极化，覆盖的液体培养的CALU-3细胞（LCC），专注于成长和培养CALU-3细胞，维持细胞已被培养成LCC的技术细节，我们提出了用于执行呼吸偏振光CALU-3细胞中的病毒感染的方法。 为了持续获得极化CALU-3 LCC，，CALU-3细胞必须小心传代培养前在Transwell小室。 CALU-3单层培养应保持在90％以下汇合，从冻结的股票应少于10次传代培养，并应定期提供新鲜培养基。一旦培养在Transwell小室内，CALU-3，LCC，必须小心处理。不规​​则的介质的变化和细胞层或板的机械或物理中断产生负面影响偏振几个小时或几天。极化评估反上皮的电阻（TEER）监测和评估的被动之间的平衡，荧光素钠的顶端和基底车厢17,18验证。一旦TEER在或1,000Ω×2厘米以上的高原，CALU-3 LCC是准备用来检查细胞对呼吸道病原体。

Transwell培养系统中的液体覆盖的培养（LCC）生长时，在图1中所示，CALU-3细胞的分化，开发独特的心尖部和基底表面。继这里描述的方法中，反式上皮电阻（TEER）CALU-3 LCC达到高原以上1,000Ω×cm 2的播种后3个星期内，它的一个例子在图2中示出。极性细胞之间形成的紧密连接，防止小分子物质心尖和基底车厢之间的被动平衡。因此，改性的荧光素钠平衡测定是用来确认CALU-3 LCC 15,17,18的偏振。由于TEER CALU-3细胞单层在LCC增加，荧光素的量，被动平衡到基底外侧隔室降低。一旦TEER的是1,000Ω×cm 2时 ，荧光素的量的平衡中基底室≤1％，如在图3中示出，因此，CALU-3 LCC被认为是完全极化时的TEER是≥1,000Ω×cm 2的 。 TEER测量的峰值的CALU-3 LCC可能会有所不同的实验，实验。然而，一旦TEER值的高原对于任何给定的实验，完全极化，未感染的的CALU 3 LCC可能是稳定的5至12周后播种。无抗性发展中的Transwell小室培养CALU-3的原因可能是由几个因素，如表1中列出。一旦TEER CALU-3 LCC以上1,000Ω×cm 2的高原，该模型是准备被用来检查气道上皮细胞对呼吸道病原体，包括呼吸道合胞病毒（RSV）。暴露RSV结果在极化文化完整性的快速下降到模拟感染的细胞（ 图4）。 图1。横截面表示的Transwell小室的培养细胞。细胞插入膜的顶端表面上生长。 <img alt="图2" fo:content-width="4in" fo:src="/files/ftp_upload/50157/50157fig2highres.jpg" src="/files/ftp_upload/50157/50157fig2.jpg" /> 图2。发展跨皮电阻（TEER）和偏振后播种的CALU-3细胞在Transwell小室。在每个时间点，TEER中位数Ω为x cm 2±SEM的32个独立的的井从一个有代表性的实验。 <img alt="图3" fo:content-width="4in" fo:src="/files/ftp_upload/50157/50157fig3highres.jpg" src="/files/ftp_upload/50157/50157fig3.jpg" /> 图3。被动式平衡<html>荧光素钠的基底仓的CALU-3细胞单层伸缩振动的抑制，细胞变得两极化，累计从四个独立的试验数据。每个数据点代表一个单独的测量。 <img alt="图4" fo:content-width="4in" fo:src="/files/ftp_upload/50157/50157fig4highres.jpg" src="/files/ftp_upload/50157/50157fig4.jpg" /> 图4。 RSV感染的偏振光CALU-3 LCC加速在单层完整性的下降。极性CALU-3细胞感染RSV-A2的MOI = 1，在第0天，监测感染后8周和极性。一个代表性的实验显示。中位数抵抗感染的井每8个独立的±SEM每个时间点的数据。 * P &lt;0.05，模拟和RSV-A2-感染的培养，为学生的t检验来确定。 办法“&gt; <tbody><tr><td> 问题 </td><td> 分辨率（S） </td></tr><tr><td>电阻是无法衡量的</td><td><ul><li>清除孔中的任何气泡</li><li>使用CALU-3传代培养的细胞已被从冷冻库存中小于10倍</li><li>不要让CALU-3细胞达到100％汇合单层传代培养</li><li>检查细胞不生长在塑料以及插入位置下方的（表明细胞可能已经插入通过毛孔生长） </li><li>全耐药性的发展，允许播种后2-3周</li><li>检查该组合物和使用的插入孔尺寸，改变推荐的聚酯插入，0.3微米的孔径，没有涂层，如有必要（见材料详情） </li><li>质量检查的电极，轻轻地打磨，清除积累的从尖端的蛋白质，并更换必要时</li><li>检查细菌生长的培养基为</li></ul&gt; </td></tr><tr><td>耐药性，但没有达到全极化（≥1000Ω×2厘米） </td><td><ul><li>允许更多的时间进行极化发展（有时可能会需要长达4周） </li><li>使用CALU-3传代培养的细胞已被从冷冻库存中小于10倍</li><li>不要让CALU-3细胞达到100％汇合单层传代培养</li><li>在Transwell文化，不断更换介质交替之间的第三和第四天后，以前的喂养</li><li>只有培养细胞的外排的板被放置在在插入</li><li>使用不同批次的Transwell小室</li></ul></td></tr><tr><td>细胞完全分化，但阻力不符合阅读</td><td><ul><li>不要破坏或堆叠板的孵化器</li><li>不要在生物安全柜中引起震动，同时Transwell小平台ES正在处理</li><li>清除孔中的任何气泡</li><li> “请勿打扰”的细胞层，吸头时不断变化的媒体或执行TEER读数</li><li>用200微升介质的顶舱量较低，可能会导致不稳定的TEER读数</li><li>质量检查的电极，轻轻地打磨，清除积累的蛋白质从尖端，必要时进行更换</li></ul></td></tr></tbody></table> 表1。故障排除可能出现的问题时，培养的CALU-3细胞在Transwell小室内。多个因素促成了全极化CALU-3细胞在液体覆盖的文化，其中最有可能的是在此表中突出显示。

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Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

本报告中的调查结果和结论是这些作者的观点，并不一定代表美国疾病控制和预防中心的意见。

建立的液体覆盖的文化偏光人类呼吸道上皮的CALU-3细胞研究呼吸系统病原宿主细胞的反应<em在体外</em

The  apical and basolateral surfaces of airway epithelial cells demonstrate  directional responses to pathogen exposure in  vivo. Thus, ideal in vitro ...

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Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

22.3K 次观看.  Centers for Disease Control and Prevention (CDC). 本报告中的调查结果和结论是这些作者的观点，并不一定代表美国疾病控制和预防中心的意见。

视频: Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells

Bacterial biofilms have been associated with a number of different human diseases, but biofilm development has generally been studied on non-living surfaces. In this paper, we describe protocols for forming Pseudomonas aeruginosa biofilms on human airway epithelial cells (CFBE cells) grown in culture. In the first method (termed the Static Co-culture Biofilm Model), P. aeruginosa is incubated with CFBE cells grown as confluent monolayers on standard tissue culture plates. Although the bacterium is quite toxic to epithelial cells, the addition of arginine delays the destruction of the monolayer long enough for biofilms to form on the CFBE cells. The second method (termed the Flow Cell Co-culture Biofilm Model), involves adaptation of a biofilm flow cell apparatus, which is often used in biofilm research, to accommodate a glass coverslip supporting a confluent monolayer of CFBE cells. This monolayer is inoculated with P. aeruginosa and a peristaltic pump then flows fresh medium across the cells. In both systems, bacterial biofilms form within 6-8 hours after inoculation. Visualization of the biofilm is enhanced by the use of P. aeruginosa strains constitutively expressing green fluorescent protein (GFP). The Static and Flow Cell Co-culture Biofilm assays are model systems for early P. aeruginosa infection of the Cystic Fibrosis (CF) lung, and these techniques allow different aspects of P. aeruginosa biofilm formation and virulence to be studied, including biofilm cytotoxicity, measurement of biofilm CFU, and staining and visualizing the biofilm.

Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells

This paper describes different methods of growing Pseudomonas aeruginosa biofilms on cultured human airway epithelial cells. These protocols can be adapted to study different aspects of biofilm formation, including visualization of the biofilm, staining of the biofilm, measuring the colony forming units (CFU) of the biofilm, and studying biofilm cytotoxicity.

共培养模型绿脓杆菌现场人类呼吸道细胞生长的生物膜

Bacterial biofilms have been associated with a number of different human diseases, but biofilm development has generally been studied on non-living ...

科研

免疫与感染

Application of a Mouse Ligated Peyer&#x2019;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed to them and occasionally to pathogens. The gut-associated lymphoid tissue (GALT) play a key role for induction of the mucosal immune response to these microbes1, 2. To initiate the mucosal immune response, the mucosal antigens must be transported from the gut lumen across the epithelial barrier into organized lymphoid follicles such as Peyer's patches. This antigen transcytosis is mediated by specialized epithelial M cells3, 4. M cells are atypical epithelial cells that actively phagocytose macromolecules and microbes. Unlike dendritic cells (DCs) and macrophages, which target antigens to lysosomes for degradation, M cells mainly transcytose the internalized antigens. This vigorous macromolecular transcytosis through M cells delivers antigen to the underlying organized lymphoid follicles and is believed to be essential for initiating antigen-specific mucosal immune responses. However, the molecular mechanisms promoting this antigen uptake by M cells are largely unknown. We have previously reported that glycoprotein 2 (Gp2), specifically expressed on the apical plasma membrane of M cells among enterocytes, serves as a transcytotic receptor for a subset of commensal and pathogenic enterobacteria, including Escherichia coli and Salmonella enterica serovar Typhimurium (S. Typhimurium), by recognizing FimH, a component of type I pili on the bacterial outer membrane 5. Here, we present a method for the application of a mouse Peyer's patch intestinal loop assay to evaluate bacterial uptake by M cells. This method is an improved version of the mouse intestinal loop assay previously described 6, 7. The improved points are as follows: 1. Isoflurane was used as an anesthetic agent. 2. Approximately 1 cm ligated intestinal loop including Peyer's patch was set up. 3. Bacteria taken up by M cells were fluorescently labeled by fluorescence labeling reagent or by overexpressing fluorescent protein such as green fluorescent protein (GFP). 4. M cells in the follicle-associated epithelium covering Peyer's patch were detected by whole-mount immunostainig with anti Gp2 antibody. 5. Fluorescent bacterial transcytosis by M cells were observed by confocal microscopic analysis. The mouse Peyer's patch intestinal loop assay could supply the answer what kind of commensal or pathogenic bacteria transcytosed by M cells, and may lead us to understand the molecular mechanism of how to stimulate mucosal immune system through M cells.

Application of a Mouse Ligated Peyer&amp;#8217;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

M cells in a specialized follicle-associated epithelium covering Peyer&#x2019;s patches play an important role for the mucosal immunosurveillance in gut-associated lymphoid tissue. Here we described the evaluation method for bacterial transcytosis by M cells in vivo. This method provides a method to understand M-cell function in the immune system.

鼠标应用结扎Peyer氏修补肠袢含量评价M细胞的细菌吸收

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed ...

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the phenotypic and functional differences between murine and human monocyte-derived macrophages. Therefore, we here describe an in vitro model to generate and study primary human macrophages. Briefly, after density gradient centrifugation of peripheral blood drawn from a forearm vein, monocytes are isolated from peripheral blood mononuclear cells using negative magnetic bead isolation. These monocytes are then cultured for six days under specific conditions to induce different types of macrophage differentiation or polarization. The model is easy to use and circumvents the problems caused by species-specific differences between mouse and man. Furthermore, it is closer to the in vivo conditions than the use of immortalized cell lines. In conclusion, the model described here is suitable to study macrophage biology, identify disease mechanisms and novel therapeutic targets. Even though not fully replacing experiments with animals or human tissues obtained post mortem, the model described here allows identification and validation of disease mechanisms and therapeutic targets that may be highly relevant to various human diseases.

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Monocyte-derived macrophages are important cells of the innate immune system. Here, we describe an easy to use in vitro model to generate these cells. Using gradient centrifugation, negative bead isolation and specific cell culture conditions, monocyte-derived macrophages can be generated for phenotypic and functional studies.

一个<em在体外</em&gt;模型研究人类巨噬细胞分化和极化的异质性

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the ...

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) infections present a broad spectrum of disease severity, ranging from mild infections to life-threatening bronchiolitis. An important part of the pathogenesis of severe disease is an enhanced immune response leading to immunopathology.	Here, we describe a protocol used to investigate the immune response of human immune cells to an HRSV infection. First, we describe methods used for culturing, purification and quantification of HRSV. Subsequently, we describe a human in vitro model in which peripheral blood mononuclear cells (PBMCs) are stimulated with live HRSV. This model system can be used to study multiple parameters that may contribute to disease severity, including the innate and adaptive immune response. These responses can be measured at the transcriptional and translational level. Moreover, viral infection of cells can easily be measured using flow cytometry. Taken together, stimulation of PBMC with live HRSV provides a fast and reproducible model system to examine mechanisms involved in HRSV-induced disease.

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) can cause severe bronchiolitis in young infants. Part of the pathogenesis of severe HRSV disease is caused by the host immune response. Stimulation of primary human immune cells with HRSV provides a fast and reproducible model system to study activation of inflammatory pathways and infection.

Human respiratory syncytial  virus (HRSV) infections present a broad spectrum of disease severity, ranging  from mild infections to life-threatening ...

Human Placental and Decidual Organ Cultures to Study Infections at the Maternal-fetal Interface

The placenta shows a large degree of interspecies anatomic variability. To best understand biology and pathophysiology of the human placenta, it is imperative to design experiments using human cells and tissues. An advantage of organ culture is maintenance of three-dimensional (3D) structural organization and extracellular matrix. The goal of the method described here is successful establishment of ex vivo human gestational tissue organ cultures and their healthy culture maintenance for 72-96 hr. The protocol details the immediate processing of research-consented, placental and decidual specimens fresh from the operating suite. These are abundant specimens that would otherwise be discarded. Detailed instructions on the sterile collection of these samples, including morphologic details on how to select appropriate tissues to establish 3D organ cultures, is provided. Placental villous and decidual tissues are microdissected into 2-3 mm3 pieces and placed separately on matrix-lined transwell filters and cultured for several days. Villous and decidual organ cultures are well suited for the study of human host-pathogen interaction. As compared to other model organisms, these human cultures are particularly advantageous to examine mechanism of infection for pathogens that demonstrate variable patterns of host specificity. As an example, we demonstrate infection of placental and decidual organ cultures with the clinically relevant, facultative intracellular bacterial pathogen Listeria monocytogenes.

A simple method to establish primary human placental (villous) and decidual organ cultures is described. Villous and decidual organ cultures are invaluable tools for studying pathogenesis at the human maternal-fetal interface. Infection with the facultative intracellular bacterium Listeria monocytogenes is demonstrated.

人胎盘及蜕膜器官培养研究在母胎界面感染

The placenta shows a large degree of interspecies anatomic variability. To best understand biology and pathophysiology of the human placenta, it is ...

Developing a Salivary Antibody Multiplex Immunoassay to Measure Human Exposure to Environmental Pathogens

The etiology and impacts of human exposure to environmental pathogens are of major concern worldwide and, thus, the ability to assess exposure and infections using cost effective, high-throughput approaches would be indispensable. This manuscript describes the development and analysis of a bead-based multiplex immunoassay capable of measuring the presence of antibodies in human saliva to multiple pathogens simultaneously. Saliva is particularly attractive in this application because it is noninvasive, cheaper and easier to collect than serum. Antigens from environmental pathogens were coupled to carboxylated microspheres (beads) and used to measure antibodies in very small volumes of human saliva samples using a bead-based, solution-phase assay. Beads were coupled with antigens from Campylobacter jejuni, Helicobacter pylori, Toxoplasma gondii, noroviruses (G I.1 and G II.4) and hepatitis A virus. To ensure that the antigens were sufficiently coupled to the beads, coupling was confirmed using species-specific, animal-derived primary capture antibodies, followed by incubation with biotinylated anti-species secondary detection antibodies and streptavidin-R-phycoerythrin reporter (SAPE). As a control to measure non-specific binding, one bead set was treated identically to the others except it was not coupled to any antigen. The antigen-coupled and control beads were then incubated with prospectively-collected human saliva samples, measured on a high throughput analyzer based on the principles of flow cytometry, and the presence of antibodies to each antigen was measured in Median Fluorescence Intensity units (MFI). This multiplex immunoassay has a number of advantages, including more data with less sample; reduced costs and labor; and the ability to customize the assay to many targets of interest. Results indicate that the salivary multiplex immunoassay may be capable of identifying previous exposures and infections, which can be especially useful in surveillance studies involving large human populations.

In the current climate of scarce resources, new technologies are emerging that allow researchers to conduct studies cheaper, faster and with more precision. Here we describe the development of a bead-based salivary antibody multiplex immunoassay to measure human exposure to multiple environmental pathogens simultaneously.

制定唾液抗体多重免疫测定来测量人体暴露于环境中的病原体

The etiology and impacts of human exposure to environmental pathogens are of major concern worldwide and, thus, the ability to assess exposure and ...

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple condensation reactions. DHDDS (dehydrodolichyl diphosphate synthase) is a eukaryotic long-chain cis-PT (forming cis double bonds from the condensation reaction) that catalyzes chain elongation of farnesyl diphosphate (FPP, an allylic diphosphate) via multiple condensations with isopentenyl diphosphate (IPP). DHDDS is of biomedical importance, as a non-conservative mutation (K42E) in the enzyme results in retinitis pigmentosa, ultimately leading to blindness. Therefore, the present protocol was developed in order to acquire large quantities of purified DHDDS, suitable for mechanistic studies. Here, the usage of protein fusion, optimized culture conditions and codon-optimization were used to allow the overexpression and purification of functionally active human DHDDS in E. coli. The described protocol is simple, cost-effective and time sparing. The homology of cis-PT among different species suggests that this protocol may be applied for other eukaryotic cis-PT as well, such as those involved in natural rubber synthesis.

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

A simple protocol for overexpression and purification of codon-optimized, human cis-prenyltransferase, under non-denaturing conditions, from Escherichia coli, is described, along with an enzymatic activity assay. This protocol can be generalized for production of other cis- prenyltransferase proteins in quantity and quality suitable for mechanistic studies.

人类的过表达和纯化<em&gt;顺</emβ-肾上腺素转移酶<em&gt;大肠杆菌</em

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple ...

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor microenvironment and the underlying mechanism is still largely unknown. The lack of a consistent and reliable source of NK cells limits the research progress of NK cell immunity. Here, we report an in vitro system that can provide high quality and quantity of bone marrow-derived murine NK cells under a feeder-free condition. More importantly, we also demonstrate that siRNA-mediated gene silencing successfully inhibits the E4bp4-dependent NK cell maturation by using this system. Thus, this novel in vitro NK cell differentiating system is a biomaterial solution for immunity research.

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Here, we present a protocol to mass-produce gene-silencing murine NK cells by using a feeder-free differentiation system for mechanistic study in vitro and in vivo.

一种新型大鼠自然杀伤细胞无饲养系统的研究体外

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor ...

Mouse- and Human-derived Primary Gastric Epithelial Monolayer Culture for the Study of Regeneration

In vitro studies of gastric wound repair typically involves the use of gastric cancer cell lines in a scratch-wound assay of cellular proliferation and migration. One critical limitation of such assays, however, is their homogenous assortment of cellular types. Repair is a complex process which demands the interaction of several cell types. Therefore, to study a culture devoid of cellular subtypes, is a concern that must be overcome if we are to understand the repair process. The gastric organoid model may alleviate this issue whereby the heterogeneous collection of cell types closely reflects that of the gastric epithelium or other native tissues in vivo. Demonstrated here is a novel, in vitro scratch-wound assay derived from human or mouse 3-dimensional organoids which can then be transferred to a gastric epithelial monolayer as either intact organoids or as a single cell suspension of dissociated organoids. The goal of the protocol is to establish organoid-derived gastric epithelial monolayers that can be used in a novel scratch-wound assay system to study gastric regeneration.

Here we describe a protocol for establishing and culturing human- and mouse-derived 3-dimensional (3D) gastric organoids, and the method for the transfer of 3D organoids to a 2-dimensional monolayer. The use of the gastric epithelial monolayer as a novel scratch-wound assay for regeneration studies is also described.

小鼠和人源性胃上皮单层培养对再生的研究

In vitro studies of gastric wound repair typically involves the use of gastric cancer cell lines in a scratch-wound assay of cellular proliferation and ...

Human Colonoid Monolayers to Study Interactions Between Pathogens, Commensals, and Host Intestinal Epithelium

Human 3-dimensional (3D) enteroid or colonoid cultures derived from crypt base stem cells are currently the most advanced ex vivo model of the intestinal epithelium. Due to their closed structures and significant supporting extracellular matrix, 3D cultures are not ideal for host-pathogen studies. Enteroids or colonoids can be grown as epithelial monolayers on permeable tissue culture membranes to allow manipulation of both luminal and basolateral cell surfaces and accompanying fluids. This enhanced luminal surface accessibility facilitates modeling bacterial-host epithelial interactions such as the mucus-degrading ability of enterohemorrhagic E. coli (EHEC) on colonic epithelium. A method for 3D culture fragmentation, monolayer seeding, and transepithelial electrical resistance (TER) measurements to monitor the progress towards confluency and differentiation are described. Colonoid monolayer differentiation yields secreted mucus that can be studied by the immunofluorescence or immunoblotting techniques. More generally, enteroid or colonoid monolayers enable a physiologically-relevant platform to evaluate specific cell populations that may be targeted by pathogenic or commensal microbiota.

Here, we present a protocol to culture human enteroid or colonoid monolayers that have intact barrier function to study host epithelial-microbiota interactions at the cellular and biochemical level.

人类舱内单聚物研究病原体、共脉和宿主肠道上皮细胞之间的相互作用

Human 3-dimensional (3D) enteroid or colonoid cultures derived from crypt base stem cells are currently the most advanced ex vivo model of the intestinal ...